Most theories on how the nervous system functions depend heavily on the existence and properties of cell to cell contacts known as synapses. For this reason, the study of synapses has been a focal point for neuroscience research for many decades.
Because of its accessibility to biochemical and electrophysiological techniques, and because of its elegant, well defined structure, the neuromuscular synapse (also known as the neuromuscular junction), which occurs at the point of nerve to muscle contact, is one of the most studied and best understood synapses. At the neuromuscular junction, the nerve cell releases a chemical neurotransmitter, acetylcholine, which binds to nicotinic acetylcholine receptor proteins located on post-synaptic muscle cells. The binding of acetylcholine results in a conformational change in the nicotinic acetylcholine receptor protein. This change is manifested by the opening of a transmembrane channel in the receptor which is permeable to cations. The resulting influx of cations depolarizes the muscle and ultimately leads to muscle contraction.
Biological and structural studies have shown that the nicotinic acetylcholine receptor in muscle is a glycoprotein composed of five subunits with the stoichiometry .alpha..alpha..beta..gamma..DELTA. (alpha-alpha-beta-gamma-delta). From these same studies, it is known that each of the subunits has a mass of about 50-60 kilodaltons and is encoded by a separate gene. In vitro reconstitution experiments have shown that this .alpha..alpha..beta..gamma..DELTA. complex is a functional receptor containing both ligand binding sites and a ligand-gated transmembrane channel.
It is now known that a variety of neurotransmitters and neurotransmitter receptors exist in the central and peripheral nervous systems. Despite this knowledge, there is still little understanding of the diversity of receptors for a particular neurotransmitter, or of how this diversity might generate different responses to a given neurotransmitter, or to other modulating ligands, in different regions of the brain. On a larger scale, there is little appreciation of how the use of a particular synapse makes it more or less efficient, or hnges in neuronal circuits might be accomplished by the modification of synapses.
An understanding of the molecular mechanisms involved in neurotransmission in the central nervous system is limited by the complexity of the system. The cells are small, have extensive processes, and often have thousands of synapses deriving from inputs from many different parts of the brain. In addition, the actual number of neurotransmitter receptors is low, making their purification difficult, even under the best of circumstances. Consequently, neither cellular nor biochemical approaches to studying neurotransmission in the central nervous system has been particularly fruitful. This is unfortunate because it is quite probable that the treatment of dementia, Alzheimer's disease and other forms of mental illness will involve modification of synaptic transmission with specific drugs.
Nicotinic acetylcholine receptors found at the vertebrate neuromuscular junction, in vertebrate sympathetic ganglia and in the vertebrate central nervous system can be distinguished pharmacologically on the basis of ligands that open or block the ion channel. For example, the elapid .alpha.-neurotoxins that block activation of nicotinic acetylcholine receptors at the neuromuscular junction do not block activation of neuronal nicotinic acetylcholine receptors found on several different cell lines.
To gain access to the neuronal acetylcholine receptors, traditional biochemical and neurophysiological methods have been abandoned in favor of the newer methods of molecular biology. More specifically, using molecular cloning techniques, complementary DNA clones were isolated which encode the acetylcholine receptor expressed in the Torpedo fish electric organ, a highly enriched source of receptor. The cDNA clones isolated from the fish electric organ were then used in nucleic acid hybridization experiments to obtain cDNA and genomic clones for the subunits of the acetylcholine receptor expressed in mouse skeletal muscle.
The availability of cDNA clones encoding muscle nicotinic receptors made it possible to extend these studies in the important direction of neuronal receptors. More specifically, based on the assumption that neuronal nicotinic receptors are evolutionarily related to muscle receptors, and that this relationship will be reflected at the genetic level by nucleotide sequence homology, the cDNA clones encoding the muscle nicotinic receptor were used to screen rat cDNA and genomic libraries for related neuronal mRNAs or genes. This method has resulted in the isolation of several neuronal cDNA clones that have significant sequence homology with the muscle acetylcholine clones.
That the neuronal nicotinic acetylcholine receptors differ from muscle nicotinic acetylcholine receptors is evidenced by the fact that neuronal receptors can be constituted from only two different gene-products (i.e., one alpha subunit and one beta subunit). This is significant since, in all experiments reported to date, muscle nicotinic acetylcholine receptors have been formed with .alpha..beta..delta..DELTA. subunits, .alpha..beta..DELTA. subunits, .alpha..beta..delta. subunits or .alpha..delta..DELTA. subunits, but not with any pairwise combinations. See Kurosaki et al., FEBS Letters 214, 253-258 (1987).
In order to further extend such studies, to provide proteins useful for assaying compounds as potential agonists or antagonists for human neuronal nicotinic acetylcholine receptors, as well as cell lines capable of expressing such proteins, we undertook to isolate and characterize clones which encode various subunits of the human neuronal nicotinic acetylcholine receptor; we further undertook to develop methods for expressing cloned human neuronal nicotinic acetylcholine receptor sequences in recombinant cell lines; and we further undertook to develop assays for identifying which of the resultant recombinant cell lines express functional neuronal nicotinic receptors.